Catabolite repression control (Crc) gene and Pseudomonas virulence

ABSTRACT

A method of screening for compounds that inhibit the virulence of  Pseudomonas  bacteria comprises the steps of: providing a culture medium comprising  Pseudomonas  bacteria; administering a test compound to said bacteria; and then detecting the presence or absence of inhibition of the catabolite repression control (Crc) protein in said bacteria, the inhibition of the Crc protein indicating said compound has antivirulence activity against  Pseudomonas  bacteria. Antivirulence compounds and the use thereof in treating  Pseudomonas  infections are also disclosed.

FIELD OF THE INVENTION

The present invention concerns methods of screening for active agentsuseful as an antibacterial agent against Pseudomonas, as well as activeagents and methods of use thereof in treating Pseudomonas.

BACKGROUND OF THE INVENTION

Pseudomonas infection is a leading cause of death in cystic fibrosis,and one of the top causes of serious hospital-acquired infections.Pseudomonas infection is particularly serious in burn victims andleukemia patients, and can cause blindness by infection in patientsafflicted with trauma to the eye through surgery or contact lenses.Further, Pseudomonas has a high intrinsic resistance to currentantibiotics. Hence, there is a need for the development of newantibiotics to treat Pseudomonas infections.

The use of antisense oligonucleotides in the treatment of bacterialinfections is known. U.S. Pat. No. 5,294,533 to J. Lupski and L. Katz(assigned to Baylor College of Medicine and Abbott Laboratories, Inc.)describes a method of interrupting the expression of a macromolecularsynthesis operon in bacteria comprising the step of binding an antisenseoligonucleotide to a single stranded DNA or to a mRNA transcribed fromthe macromolecular synthesis operon. The antisense oligonucleotide canbe either sequence specific to a unique intergenic sequence or asequence specific to a bacterial homologous sequence. By interruptingthe expression of the macromolecular synthesis, it is said thatbacterial infections can be treated.

U.S. Pat. No. 6,060,241 to I. Corthesy-Theulaz (assigned to KietaHolding SA) describes a poly-3-hydroxybutyrate metabolic pathwayessential for Helicobacter pylori survival in a host. A Helicobacterpylori Coenzyme A transferase (Hp CoA-t), thiolase and PHB synthase, aswell as methods for their preparation and use are provided.Pharmaceutical compositions containing Hp CoA-t protein fragments,antisense nucleic acids or other inhibitors of Hp CoA-t, thiolase andPHB synthase, as well as methods for their use in the treatment of sometypes of gastric disease are also described. This reference is notconcerned with Pseudomonas.

PCT Application WO98/03533A1 to A. Arrow et al. (assigned to Oligos Etc.and Oligos Therapeutics Inc.) describes the general therapeutic use ofnuclease resistant oligonucleotides for treating animals having aninfection caused by a pathogenic bacterium. The method is a general oneand involves the integration of (1) methods for selecting the correctoligonucleotide, (2) synthesis and purification of nuclease resistantoligonucleotides, and (3) methods for in vitro analysis of potentialantimicrobial oligonucleotides.

There remains a need for new ways to screen for antibiotics effectiveagainst Pseudomonas, along with compounds and methods of treatingPseudomonas infections.

SUMMARY OF THE INVENTION

The present invention provides a method of screening for compounds thatinhibit the virulence of Pseudomonas bacteria. The method comprises thesteps of: providing a culture medium comprising Pseudomonas bacteria;administering a test compound to said bacteria; and then detecting thepresence or absence of inhibition of the catabolite repression control(Crc) protein in said bacteria, the inhibition of the Crc proteinindicating said compound has antivirulence activity against Pseudomonasbacteria.

A second aspect of the present invention is an antivirulence compound(for example, an antisense oligonucleotide) that inhibits expression oractivity of the Crc protein in a Pseudomonas bacteria. Such compoundsare useful as antivirulence compounds. When such compounds are antisenseoligonucleotides they are preferably from 8 to 25, 40 or 80 nucleotidesin length, and preferably are nuclease resistant.

A third aspect of the present invention is an antivirulence compound asdescribed above in a pharmaceutically acceptable carrier.

A fourth further aspect of the present invention is a method ofinhibiting the virulence of Pseudomonas bacteria, comprisingadministering to Pseudomonas bacteria an antivirulence compound asdescribed above in an effective antivirulence amount. The administeringstep may be carried out in vitro, for example in drug testing orscreening studies, or may be carried out in vivo in the treatment of asubject.

A further aspect of the present invention is a method of treatingPseudomonas infection in a subject in need thereof, comprisingadministering to said subject an antivirulence compound as describedabove in an amount effective to treat said Pseudomonas infection.

A still further aspect of the present invention is the use of anantivirulence compound as described above for the preparation of amedicament for carrying out a method as described above.

The foregoing and other objects and aspects of the present invention areexplained in greater detail in the specification set forth below.

DETAILED DESCRIPTION

The term “Pseudomonas” as used herein refers to any type of Pseudomonasbacteria, including but not limited to Pseudomonas aeruginosa,Pseudomonas putida, Pseudomonas fluorescens, and Pseudomonasmultivorans. Particularly preferred for carrying out the presentinvention is Pseudomonas aeruginosa.

The terms “antivirulence” and “antivirulent” as used herein refers tothe activity of a compound in reducing, at least in part, the degree ofpathogenicity of a microorganism, as indicated by fatality rate ofinfected hosts infected with that microorganism and/or the ability ofthat microorganism to invade the tissues of an infected host.

The term “compound” as used herein refers to a peptide, peptidomimetic,organic, or other chemical molecule, and also refers to a nucleic acidmolecule or chemical derivative thereof.

The term “oligonucleotide” herein includes naturally occurring, andmodified nucleotides linked together by naturally occurring, andnon-naturally occurring oligonucleotide linkages. Oligonucleotides areusually a polynucleotide subset with 200 bases or fewer in length.Preferably oligonucleotides are minimally 10 to 60 bases in length andmost preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases inminimal length. Oligonucleotides are usually single-stranded, e.g. forprobes; although oligonucleotides may be double-stranded, e.g. for usein the construction of a gene mutant. Oligonucleotides of the inventioncan be either sense or antisense oligonucleotides. The term “naturallyoccurring nucleotides” referred to herein includes deoxyribonucleotidesand ribonucleotides. The term “modified nucleotides” referred to hereinincludes nucleotides with modified or substituted sugar groups and thelike. The term “oligonucleotide linkages” referred to herein includesoligonucleotides linkages such as phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phoshoraniladate, phosphoroamidate, and the like. An oligonucleotide caninclude a label for detection, if desired. Peptide nucleic acids (PNAs)are a specific example of oligonucleotides included within the term“oligonucleotide”. Examples of peptide nucleic acid structures (in theappropriate sequence as given below), that may be used to carry out thepresent invention include, but are not limited to, those described inU.S. Pat. Nos. 5,986,053; 6,133,444; 6,107,470; 6,015,887; 6,015,710;5,846,010; 5,773,571; 5,672,584; 5,539,083; and 5,539,082 (applicantsspecifically intend that the disclosures of all United States patentreferences cited herein are to be incorporated by reference herein intheir entirety).

Subjects with which the present invention is concerned include bothhuman subjects and animal subjects (particularly mammalian subjects suchas dogs, cats, horses, cattle) for veterinary purposes.

1. Screening Methods.

Test compounds, including combinatorial libraries of such compounds,that may be screened for activity by the methods of the invention are,in general, small organic compounds (i.e., non-oligomers), oligomers, orcombinations thereof. Compounds which exhibit activity in these methodsare referred to as “active compounds” below.

Small organic compounds (or “non-oligomers”) include a wide variety oforganic molecules, such as heterocyclics, aromatics, alicyclics,aliphatics and combinations thereof, comprising steroids, antibiotics,enzyme inhibitors, ligands, hormones, drugs, alkaloids, opioids,terpenes, porphyrins, toxins, catalysts, as well as combinationsthereof. Libraries of such compounds are available, examples includingbenzodiazepine libraries as described in U.S. Pat. No. 5,288,514;phosphonate ester libraries as described in U.S. Patent No. 5,420,328,pyrrolidine libraries as described in U.S. Pat. Nos. 5,525.735 and5,525,734, and diketopiperazine and diketomorpholine libraries asdescribed in U.S. Pat. No. 5,817,751.

Oligomers include oligopeptides, oligonucleotides, oligosaccharides,polylipids, polyesters, polyamides, polyurethanes, polyureas,polyethers, and poly (phosphorus derivatives), e.g. phosphates,phosphonates, phosphoramides, phosphonamides, phosphites,phosphinamides, etc., poly (sulfur derivatives) e.g., sulfones,sulfonates, sulfites, sulfonamides, sulfenamides, etc., where for thephosphorous and sulfur derivatives the indicated heteroatom for the mostpart will be bonded to C,H,N,O or S, and combinations thereof. Sucholigomers may be obtained from combinatorial libraries in accordancewith known techniques, or may be designed to have an antisense sequencebased upon the known sequence of the Crc gene, such as assigned GenBankaccession number L12038 (C. MacGregor et al., J. Bacteriol. 178,5627-5635 (1996). Examples of such oligonucleotides include, but are notlimited to, those having the sequence given herein as SEQ ID NO:1, SEQID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

As noted above, a method of screening for compounds that inhibit thevirulence of Pseudomonas bacteria comprises first providing a culturemedium comprising Pseudomonas bacteria and administering a test compoundto said bacteria. The bacterial culture can be provided in any suitableform, such as in a petri dish on a growth medium, and the test compoundcan be administered to the bacterial culture in any way, such as bydiluting the test compound and applying it to colonies of bacteria on apetri dish; mixing bacteria in a solution containing the test compoundand then applying those bacteria to a culture medium in a petri dish;etc.

The step of detecting the presence or absence of inhibition of thecatabolite repression control (Crc) protein in the bacteria can becarried out directly (e.g., by the use of oligonucleotide probes for Crcgene expression activity; by isolation and quantification of expressedCrc protein) or indirectly (e.g., by observation of responses mediatedby the Crc gene and encoded protein). It will be noted that thedetecting step is a selective one: that is, the test compound is notapplied in an antibacterial amount to the bacteria, as this wouldprevent the identification of compounds that selectively inhibit the Crcprotein.

A particularly suitable screening assay is one in which fluoroacetamideis used in the bacterial culture. Fluoroacetamide is ordinarilymetabolized to fluoroacetic acid by amidase in Pseudomonas bacteria,with the fluoroacetic acid being toxic to the bacteria. Amidase operonrepressors, which are mediated by crc, reduce the amount of amidase inthe cells and thus protect the cells from fluoroacetic acid toxicity.Any suitable amidase operon repressor can be used, including Krebs cycleintermediates and acetate, with succinic acid particularly preferred.Thus, test compounds that inhibit the crc protein can be detected byadding an amidase operon repressor to the culture medium, addingfluoroacetamide to the culture medium, and administering the testcompound to the bacteria. Detection of crc inhibition can then becarried out by detecting the poisoning of said bacteria (e.g., death ofbacteria, inibition of bacterial growth or function, etc.) by thefluoroacetamide (specifically, by its toxic degredation productfluoroacetic acid). The poisoning of the bacteria by the fluoroacetamideindicates the test compound has antivirulence activity againstPseudomonas bacteria.

2. Active Compounds and Pharmaceutical Formulations.

Active compounds (or “antivirulence compounds”) of the present inventionare compounds that exhibit antivirulence activity in the screeningprocedures described above. Such compounds may be oligomers (includingantisense oligonucleotides) or nonoligomers.

Antisense oligonucleotides of the invention may comprise any polymericcompound capable of specifically binding to a target polynucleotide byway of a regular pattern of monomer-to-nucleoside interactions, such asWatson-Crick type of base pairing, Hoogsteen or reverse Hoogsteen typesof base pairing, or the like. Antisense compounds of the invention mayalso contain pendent groups or moieties, either as part of or separatefrom the basic repeat unit of the polymer, to enhance specificity,nuclease resistance, delivery, or other property related to efficacy,e.g. cholesterol moieties, duplex intercalators such as acridine,poly-L-lysine, “end capping” with one or more nuclease-resistant linkagegroups such as phosphorothioate, and the like. Thus, numerous different“backbone” chemistries, including the peptide nucleic acid chemistries,can be used for the oligonucleotides, as long as the desired ornecessary sequence is incorporated into the particular molecule used.

Antisense compounds of the invention include the pharmaceuticallyacceptable salts thereof, including those of alkaline earths, e.g.sodium or magnesium, ammonium or NX₄+, wherein X is C₁-C₄ alkyl. Otherpharmaceutically acceptable salts include organic carboxylic acids suchas acetic, lactic, tartaric, malic, isethionic, lactobionic, andsuccinic acids; organic sulfonic acids such as methanesulfonic,ethanesulfonic, and benzenesulfonic; and inorganic acids such ashydrochloric, sulfuric, phosphoric, and sulfamic acids. Pharmaceuticallyacceptable salts of a compound having a hydroxyl group include the anionof such compound in combination with a suitable cation such as Na+,NH₄+, or the like.

Preferably, nuclease resistance is conferred on the antisense compoundsof the invention by providing nuclease-resistant internucleosidiclinkages. Many such linkages are known in the art, e.g.phosphorothioate: Zon and Geiser, Anti-Cancer Drug Design, 6: 539-568(1991); Stec et al, U.S. Pat. No. 5,151,510; Hirschbein, U.S. Pat. No.5,166,387; Bergot, U.S. Pat. No. 5,183,885; phosphorodithioates:Marshall et al, Science, 259:1564-1570 (1993); Carathers and Nielsen,International application PCT/US89/02293; phosphoramidates, e.g.—OP(═O)(NR¹R²)—O— with R¹ and R² hydrogen or C₁-C₃ alkyl: Jager et al,Biochemistry, 27:7237-7246 (1988); Froehler et al, Internationalapplication PCT/US90/03138; peptide nucleic acids: Nielsen et al,Anti-cancer Drug Design, 8: 53-63 (1993), International applicationPCT/EP92/O1220; methylphosphonates: Miller et al, U.S. Pat. No.4,507,433; Ts'o et al, U.S. Pat. No. 4,469,863; Miller et al, U.S. Pat.No. 4,757,055; and P-chiral linkages of various types, especiallyphosphorothioates, Stec et al, European patent application 92301950.9and Lesnikowski, Bioorganic Chemistry, 21:127-155 (1993). Additionalnuclease linkages include phosphoroselenoate, phosphorodiselenoate,phosphoroanilothioate, phosphoranilidate, alkylphosphotriester such asmethyl- and ethylphosphotriester, carbonate such as carboxymethyl ester,carbamate, morpholino carbamate, 3′-thioformacetal, silyl such asdialkyl (C₁-C₆)— or diphenylsilyl, sulfamate ester, and the like. Suchlinkages and methods for introducing them into oligonucleotides aredescribed in many references, e.g. reviewed generally by Peyman andUlmann (cited above); Milligan et al (cited above); Matteucci et al,International application PCT/US91/06855. Preferably, phosphorus analogsof the phosphodiester linkage are employed in the compounds of theinvention, such as phosphorothioate, phosphorodithioate,phosphoramidate, or methylphosphonate. More preferably, phosphorothioateis employed as the nuclease resistant linkage. It its understood that inaddition to the preferred linkage groups, compounds of the invention maycomprise additional modifications, e.g. boronated bases, Spielvogel etal, U.S. Pat. No. 5,130,302; cholesterol moieties, Shea et al, NucleicAcids Research, 18:3777-3783 (1990) or Letsinger et al, Proc. Natl.Acad. Sci., 86:6553-6556 (1989); 5-propenyl modification of pyrimidines,Froehler et al, Tetrahedron Lett., 33: 5307-5310 (1992); and the like.

Other examples of antisense oligonucleotides (i.e., chemical backbonestructures) that may be used to carry out the present invention includethose described in U.S. Pat. No. 6,015,866 to Arrow and U.S. Pat. No.5,989,912 to Arrow.

Preferably, antisense compounds of the invention are synthesized byconventional means on commercially available automated DNA synthesizers,e.g. an Applied Biosystems (Foster City, Calif.) model 380B, 392 or 394DNA/RNA synthesizer. Preferably, phosphoramidite chemistry is employed,e.g. as disclosed in the following references: Beaucage and Iyer,Tetrahedron, 48:2223-2311 (1992); Molko et al, U.S. Pat. No. 4,980,460;Koster et al, U.S. Pat. No. 4,725,677; Caruthers et al, U.S. Pat. Nos.4,415,732;4,458,066; and 4,973,679; and the like.

In embodiments where triplex formation is desired, there are constraintson the selection of target sequences. Generally, third strandassociation via Hoogsteen type of binding is most stable alonghomopyrimidine-homopurine tracks in a double stranded target. Usually,base triplets form in T-A*T or C-G*C motifs (where “-” indicatesWatson-Crick pairing and “*” indicates Hoogsteen type of binding);however, other motifs are also possible. For example, Hoogsteen basepairing permits parallel and antiparallel orientations between the thirdstrand (the Hoogsteen strand) and the purine-rich strand of the duplexto which the third strand binds, depending on conditions and thecomposition of the strands. There is extensive guidance in theliterature for selecting appropriate sequences, orientation, conditions,nucleoside type (e.g. whether ribose or deoxyribose nucleosides areemployed), base modifications (e.g. methylated cytosine, and the like)in order to maximize, or otherwise regulate, triplex stability asdesired in particular embodiments, e.g. Roberts et al, Proc. Natl. Acad.Sci., 88:9397-9401 (1991); Roberts et al, Science, 258:1463-1466 (1992);Distefano et al, Proc. Natl. Acad. Sci., 90:1179-1183 (1993); Mergny etal, Biochemistry, A 30:9791-9798 (1991); Cheng et al, I. Am. Chem. Soc.,114:4465-4474 (1992); Beal and Dervan, Nucleic Acids Research,20:2773-2776 (1992); Beal and Dervan, 1. Am. Chem. Soc., 114:4976-4982(1992); Giovannangeli et al, Proc. Natl. Acad. Sci., 89: 8631-8635(1992); Moser and Dervan, Science, 238:645-650 (1987); McShan et al, J.Biol. Chem., 267:5712-5721 (1992); Yoon et al, Proc. Natl. Acad. Sci.,89:3840-3844 (1992); Blume et al, Nucleic Acids Research, 20:1777-1784(1992); and the like.

The length of the oligonucleotide moieties is sufficiently large toensure that specific binding will take place only at the desired targetpolynucleotide and not at other fortuitous sites, as explained in manyreferences, e.g. Rosenberg et al, International applicationPCT/US92/05305; or Szostak et al, Meth. Enzymol. 68:419-429 (1979). Theupper range of the length is determined by several factors, includingthe inconvenience and expense of synthesizing and purifying oligomersgreater than about 30-40 nucleotides in length, the greater tolerance oflonger oligonucleotides for mismatches than shorter oligonucleotides,whether modifications to enhance binding or specificity are present,whether duplex or triplex binding is desired, and the like. Usually,antisense compounds of the invention have lengths in the range of about12 to 60 nucleotides. More preferably, antisense compounds of theinvention have lengths in the range of about 15 to 40 nucleotides; andmost preferably, they have lengths in the range of about 18 to 30nucleotides.

The antisense oligonucleotides of the invention can be synthesized byany of the known chemical oligonucleotide synthesis methods. Suchmethods are generally described, for example, in Winnacker, From Genesto Clones: Introduction to Gene Technology. VCH Verlagsgesellschaft mbH(H., Ibelgaufts trans. 1987). Any of the known methods ofoligonucleotide synthesis can be utilized in preparing the instantantisense oligonucleotides. The antisense oligonucleotides are mostadvantageously prepared by utilizing any of the commercially available,automated nucleic acid synthesizers. The device utilized to prepare theoligonucleotides described herein, the Applied Biosystems 380B DNASynthesizer, utilizes β-cyanoethyl phosphoramidite chemistry.

Oligonucleotides complementary to and hybridizable with any portion ofthe Crc gene mRNA transcript are, in principle, effective for inhibitingtranslation of the transcript, and capable of inducing the effectsherein described. Translation is most effectively inhibited by blockingthe mRNA at a site at or near the initiation codon. Thus,oligonucleotides complementary to the 5′-terminal region of the Hp CoA-tor thiolase mRNA transcript are preferred. Secondary or tertiarystructure which might interfere with hybridization is minimal in thisregion. The antisense oligonucleotide is preferably directed to a siteat or near the ATG initiation codon for protein synthesis.Oligonucleotides complementary to a portion of the Hp CoA-t or thiolasemRNA including the initiation codon are preferred. While antisenseoligomers complementary to the 5′-terminal region of the Hp CoA-t orthiolase transcript are preferred, particularly the region including theinitiation codon, it should be appreciated that useful antisenseoligomers are not limited to those complementary to the sequences foundin the translated portion of the mRNA transcript, but also includesoligomers complementary to nucleotide sequences contained in, orextending into, the 5′- and 3′-untranslated regions.

Preferably, the thermal stability of the antisense oligonucleotides ofthe invention are determined by way of melting, or strand dissociation,curves. The temperature of fifty percent strand dissociation is taken asthe melting temperature, T_(m), which, in turn, provides a convenientmeasure of stability. T_(m) measurements are typically carried out in asaline solution at neutral pH with target and antisense oligonucleotideconcentrations at between about 1.0-2.0 μM. Typical conditions are asfollows: 150 mM NaCl and 10 mM MgCl₂ in a 10 mM sodium phosphate buffer(pH 7.0) or in a 10 mM Tris-HCl buffer (pH 7.0); or like conditions.Data for melting curves are accumulated by heating a sample of theantisense oligonucleotide/target polynucleotide complex from roomtemperature to about 85-90° C. As the temperature of the sampleincreases, absorbance of 260 nm light is monitored at 1° C. intervals,e.g. using a Cary (Australia) model 1E or a Hewlett-Packard (Palo Alto,Calif.) model HP 8459 UV/VIS spectrophotometer and model HP 89100Atemperature controller, or like instruments. Such techniques provide aconvenient means for measuring and comparing the binding strengths ofantisense oligonucleotides of different lengths and compositions.

In yet another aspect, a pharmaceutical composition comprises a compoundthat inhibits the activity of the crc protein. Such compound can beidentified by any of the methods described above.

Pharmaceutical compositions of the invention include a pharmaceuticalcarrier that may contain a variety of components that provide a varietyof functions, including regulation of drug concentration, regulation ofsolubility, chemical stabilization, regulation of viscosity, absorptionenhancement, regulation of pH, and the like. For example, in watersoluble formulations the pharmaceutical composition preferably includesa buffer such as a phosphate buffer, or other organic acid salt,preferably at a pH of between about 7 and 8. For formulations containingweakly soluble antisense compounds, microemulsions may be employed, forexample by using a nonionic surfactant such as Tween 80 in an amount of0.04-0.05% (w/v), to increase solubility. Other components may includeantioxidants, such as ascorbic acid, hydrophilic polymers, such as,monosaccharides, disaccharides, and other carbohydrates includingcellulose or its derivatives, dextrins, chelating agents, such as EDTA,and like components well known to those in the pharmaceutical sciences,e.g. Remington's Pharmaceutical Science, latest edition (Mack PublishingCompany, Easton, Pa.).

An effective amount of oligonucleotide for particular applicationsdepends on several factors, including the chemical nature of theantisense oligonucleotide, the disorder being treated, the method ofadministration, and the like. Preferably, an effective amount willprovide a concentration of oligonucleotide of between about 1 to 100 μMat the target polynucleotide; and more preferably, an effective amountwill provide a concentration of antisense oligonucleotide of betweenabout 1 to 10 μM at the target polynucleotide.

Depending on the structural and stability characteristics of the activecompound, the per unit dosage and precise formulation of thepharmaceutical composition may vary. Typically, such compound would beadministered orally at a dose ranging from 0.08 mg to 5 g daily,preferably between 0.2 mg to 0.2 g daily, most preferably between 0.8 mgto 100 mg daily. Preferably the compound would be administered multipletimes per day and can be administered in a single dose, although this isless preferred. Typically, the drug delivery vehicle, whether liquid,gel, tablet, or another vehicle, will permit effective release of thecompound at the site of infection. The drug delivery vehicle can providefor either immediate release or systematic release over time at the siteof infection. The inhibitor compound can be administered parenterally,such as intravenously, but this is less preferred. The compound can alsobe administered prophylactically to prevent the onset of diseaseassociated with bacterial infection.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder such as polyvinylpyrrolidone, gum tragacanth,acacia, sucrose, corn starch or gelatin; an excipient such as calciumphosphate, sodium citrate and calcium carbonate; a disintegrating agentsuch as corn starch, potato starch, tapioca starch, certain complexsilicates, alginic acid and the like; a lubricant such as sodium laurylsulfate, talc and magnesium stearate; a sweetening agent such assucrose, lactose or saccharin; or a flavoring agent such as peppermint,oil of wintergreen or cherry flavoring. Solid compositions of a similartype are also employed as fillers in soft and hard-filled gelatincapsules; preferred materials in this connection also include lactose ormilk sugar as well as high molecular weight polyethylene glycols. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye, flavoring such as cherry or orange flavor,emulsifying agents and/or suspending agents, as well as such diluents aswater, ethanol, propylene glycol, glycerin and various like combinationsthereof. Of course, any material used in preparing any dosage unit formshould be pharmaceutically pure and substantially non-toxic in theamounts employed.

The present invention is explained in greater detail in the followingnon-limiting Examples.

EXAMPLE 1 Virulence of crc+ and crc− Pseudomonas aeruginosa in a MouseBurn Model

A wild-type strain of P. aeruginosa designated PAO0001, a crc pointmutation strain of P. aeruginosa designated PAO8007, and a crc knock-outstrain of P. aeruginosa designated PAO8020, were tested in a burnedmouse model of bacterial virulence. 200 cells of the indicated strainwere injected into the burned skin wound of the animals. At 48 hourspost burn/infection death was observed in four of the five mice infectedwith PAO0001. Large numbers of PAO0001 were recovered from skin, blood,liver, spleen and other organs at death. This is similar to themortality found in other strains of P. aeruginosa.

In contrast, only one of five animals infected with PAO8007 was dead 48hours after burn and infection (again with 200 bacterial cells), and allfour remaining animals were still alive 7 days after burn and infection.Similar results were seen with animals infected with PAO8020.

The foregoing study was repeated with PAO8007 and PAO8020, but ten timesthe usual amount of bacteria was injected into the burn wound (i.e.,2,000 bacterial cells per animal). Essentially the same results wereseen.

EXAMPLE 2 Fluoroacetamide Plate Assay for Crc

This assay (described in O'Toole et al., J. Bacteriol. 182, 425-431(2000)) takes advantage of the Crc mediated repression of the amidaseoperon of P. aeruginosa by organic acids. Amidase hydrolyzes thefluoroacetamide into the toxic intermediate fluoroacetic acid. The stepsare as follows:

-   -   (1) streak bacteria onto a BSM minimal (W Lynch and M. Franklin,        Arch. Microbiol. 118, 133-140 (1978)) plate containing 40 mM        succinate and grow overnight at 37 degrees centigrade.    -   (2) Restreak bacteria onto BSM minimal containing 40 mM        succinate and 2.5 mg/ml fluoroacetamide (FAA) (Aldrich        12,834-1). Incubate about 36 hours at 37 degrees. In crc+        strains, succinate represses the expression of amidase and        allows for growth in the presence of FAA. Catabolite repression        control-negative (crc−) mutant strains express amidase which        converts FAA to the toxic intermediate fluoroacetate, hence the        mutants do not grow.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1-9. (Canceled)
 10. An antisense oligonucleotide that inhibitsexpression of a catabolite repressor control (Crc) protein in aPseudomonas bacteria, wherein said antisense oligonucleotide comprisesfrom 8 to 80 nucleotides and is nuclease resistant.
 11. The antisenseoligonucleotide according to claim 10 in a pharmaceutically acceptablecarrier.
 12. A method of inhibiting the virulence of Pseudomonasbacteria, comprising administering to Pseudomonas bacteria an antisenseoligonucleotide according to claim 10 in an effective antivirulenceamount.
 13. A method according to claim 12, wherein said administeringstep is carried out in vitro.
 14. A method according to claim 12,wherein said administering step is carried out in vivo.
 15. A method oftreating Pseudomonas infection in a subject in need thereof, comprisingadministering to said subject an antisense oligonucleotide according toclaim 10 in an amount effective to treat said Pseudomonas infection.